Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry

• Published in: PLoS pathogens 2014-07, Vol.10 (7), p.e1004295
• Main Authors: Eshraghi, Aria, Dixon, Shandee D, Tamilselvam, Batcha, Kim, Emily Jin-Kyung, Gargi, Amandeep, Kulik, Julia C, Damoiseaux, Robert, Blanke, Steven R, Bradley, Kenneth A
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Animals; Bacteria; Bacterial Toxins - pharmacology; Biology and Life Sciences; Blotting, Western; Cell cycle; Cell Membrane - metabolism; Chancroid - metabolism; Chancroid - microbiology; Chancroid - pathology; CHO Cells; Cricetinae; Cricetulus; Deoxyribonucleic acid; DNA; DNA damage; Drug dosages; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum-Associated Degradation - drug effects; Experiments; Flow cytometry; Gene Expression Regulation - drug effects; Golgi Apparatus - metabolism; Haemophilus ducreyi - growth & development; Haemophilus ducreyi - pathogenicity; Health aspects; HeLa Cells; Humans; Immunoprecipitation; Immunosuppressive Agents - pharmacology; Medicine and Health Sciences; Membrane Proteins - genetics; Membrane Proteins - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Protein Transport - drug effects; Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Toxins; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Virulence (Microbiology)
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1004295

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.